Cellulose, (1-4)-linked-xcex2-D-glucan, is a major structural component of the cell walls of higher plants (Delmer, D. P., and Amor, Y., Plant Cell 7:987-1000 (1995)). Some microorganisms also produce unbranched (1-4)-linked-xcex2-D-glucan, named microbial cellulose (MC) (Schramm, M. and Hestrin, S., Biochem. J., 56:163-166 (1954); Carr, J. G., Nature (London), 182:265-266 (1958) and Canale-Parola, E. and Wolfe, R. S., Biochim. Biophys. Acta. 82:403-405 (1964)). Cellulose is important industrially, for example, in the production of paper. Cellulose can be chemically detergents, varnishes, adhesives and for gelling or thickening of food stuffs or pharmaceuticals, depending on the extent of etherification.
Structurally related polysaccharides, such as chitin and chitosan, are also found in the biosphere. Chitin occurs as a major cuticular or skeletal component in all arthropods, in some invertebrates, and in the cell walls of some fungi. Chitin is a polysaccharide of high molecular weight and consists of unbranched chains of (1-4)-linked 2- acetamino-2-deoxy-xcex2-D-glucose residues (Hackman, R. H. and Goldberg, M., Carbohydr. Res. 38:35-45 (1974)). Because of its abundance as a waste material from the canning food industry from crab, shrimp and lobster, chitin is an attractive starting material for the production of chitosan. Chitosan is the fully or partially deacetylated form of chitin (Anthosen, M. W., et al., Carbohydr. Polym. 22:193-201 (1993)). It contains xcex2-(1-4)-linked 2-amino-2-deoxy-xcex2-D-glucopyranose and 2-acetamido-2-deoxy-xcex2-D-glucopyranose residues (Hirano, S., et al., Carbohydr. Res. 47:315-320(1976)). Chitosan is found in the cell walls of some fungi such as Mucor rouxii (Bartnicki-Garcia, S. and Nickerson, W. J., J. Bacteriol. 84:841-858 (1962)). However, like cellulose, chitin is generally insoluble in water and in most conventional solvent systems. Furthermore, the starting material, chitin, is easily degraded in the presence of acid.
Commercially, chitosan is derived by the chemical deacetylation of chitin from waste crustacean exoskeletons with strong alkali. This harsh conversion process, as well as variability in source material, leads to inconsistent physicochemical characteristics (Arcidiacono, S. and Kaplan, D. L. Biotechnol. Bioeng., 39:281-286 (1992).). The purification of chitosan derived from the cell wall of some fungi also requires strong alkaline treatment with heat, which leads to inconsistent material (White, S. A., et al., Environ. Microbiol., 38:323-328 (1979); Arcidiacono, S. and Kaplan, D. L. Biotechnol. Bioeng., 39:281-286 (1992)).
Glucose-rich polysaccharides such as cellulose and curdlan have been post-biosynthetically modified by nonspecific chemical means to change physical properties (Yamamoto, I. et al., Carbohydr. Polym., 14:53-63 (1991); Osawa, Z., et al., Carbohydr. Polym., 21:283-288 (1993)). For example, chemically modified cellulose and curdlan exhibited strong antiviral activity in vitro (Yamamoto, I. et al., Carbohydr. Polym., 14:53-63 (1991); Osawa, Z. et al., Carbohydr. Polym., 21:283-288 (1993)). Selective chemical modification of polysaccharides under homogeneous conditions also has been reported (Roesser, D. S. et al., Macromol., 29:1-9 (1996)). However, it is extremely difficult to regiospecifically modify cellulose in the secondary hydroxyl position, to chemically generate glucosamine or N-acetylglucosamine, for example. Disadvantages of these synthetic approaches as well as purification of chitin from crustacean exoskeleton and plant and fungi cell walls include low yields, side reactions, the use of toxic solvents, and purification requirements.
Biosynthesis of polysaccharides has traditionally been studied using unmodified simple sugars such as glucose and sucrose, or complex carbon sources such as wheat gluten and molasses (Kaplan, D. L. et al., xe2x80x9cBiosynthetic Polysaccharides In Biomedical polymers: designed-to-degrade systems,xe2x80x9d edited by S. W. Shalaby, Hanser Publishers, New York. pp. 189-212 (1994)). Alternatively, microbial mutants have been used to manipulate biopolymer molecular weight, yield, and main chain or branch composition (Thorne, L., et al., J. Bacteriol. 169:3593-3600 (1987); Hassler, R. A. and Doherty, D. H., Biotechnol. Prog. 6:182-187 (1990)). Yet polysaccharides have not been well studied with respect to the incorporation of modified or non-native building blocks, unlike the extensive work with proteins for the incorporation of unnatural amino acids (Chung, H., et al., Science 259:806-809 (1993)), and bacterial polyesters with incorporation of a wide range of novel monomers (Brandl, H., et al., xe2x80x9cPlastics from Bacteria and for Bacteria: Poly(xcex2-Hydroxyalkanoates) as Natural, Biocompatible and Biodegradable Polyesters In Advances in Biochemical Engineering/Biotechnology,xe2x80x9d Vol. 41, edited by T. K. Ghose and A. Fiechter. Springer, Berlin. pp.77 (1990); Steinbxc3xcchel, A., xe2x80x9cPolyhydroxyalkanoic Acids In Biomaterials: Novel Materials from Biological Sources, edited by D. Byrom, Stockton Press, New York. pp. 123 (1991); Gross, R. A., xe2x80x9cBacterial Polyesters: Structural Variability in Microbial Synthesis In Biomedical Polymers: Designed-to-Degrade Systems, edited by S. W. Shalaby. Hanser Publishers, New York, pp. 173-188 (1994)).
Direct incorporation of glucose-related sugar monomers, 3-O-methyl-D-glucose (3-O-methylglucose) and 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine), into the main chain of biosynthesized curdlan has been reported (Lee, J. W. et al., Can. J. Microbiol. 43:149-156 (1997)). In related studies, direct incorporation of specific fatty acid pendent groups on a main chain polysaccharide such as emulsan has been demonstrated (Gorkovenko, A. et al., Proc. Am. Chem. Soc. Div. Poly. Sci. Eng., 72:92-94 (1995); Gorkovenko, A. et al., Can. J. Microbiol., 43:384-390 (1997); Zhang, J., et al., Int. J. Biol. Macromol., 20:9-21 (1997)).
Bacterial cellulose containing a limited amount of N-acetylglucosamine has been described, however, the method to produce the copolymer required serial adapation of the bacteria in N-acetylglucosaime containing medium (Ogawa and Tokura, Carbohydrate Polymers, 19:171-178 (1992)). Furthermore, the copolymer produced only contained the glucose analog N-acetyglucosamine, at a mole percentage in liquid culture no greater than 4.5% (Ogawa and Tokura). Incorporation of up to 6.3% of N-acetylglucosamine has been achieved when bacteria were serially adapted to culture in N-acetylglucosamine and cultured in the presence of phosphorylated chitin (Shirai et al., Int. J. Biol. Macromol., 16:297-300 (1994)).
Therefore, a method is needed to produce polysaccharides comprising useful glucose analogs such as glucosamine and N-acetylglucosamine that does not require harsh extraction protocols and such that variable levels of glucose analog incorporation can be achieved. Further, a method for the production of polysaccharides comprising glucose and glucose analogs other than N-acetylglucosamine is needed.
In the present invention, microbially produced polysaccharide copolymers are provided. Specifically, glucose analogs such as aminosugars are incorporated into polysaccharides produced as exopolymers (also referred to herein as copolymers or terpolymers) by A. xylinum. Examples of aminosugars are glucosamine and N-acetylglucosamine.
Polymer blends of cellulose-chitin and cellulose-chitosan have been reported; however, the availability of copolymers with the level of incorporation of these monomers as provided herein is novel and expected to result in new properties as well as enhanced control over structural features of the polysaccharide. Furthermore, the method of the present invention does not require adaptation of the polymer producing microbe to growth in glucose analog containing medium. Copolymers described herein possess new properties such as unique solubility behavior. In addition, direct formation of fibers by the bacteria are useful in the study of crystallinity and mechanical properties of the copolymers.
An advantage of bacterially produced cellulose and the copolymers of the present invention is that these molecules are produced by the bacteria in a commercially useful fiber form. Other sources of cellulose and chemically modified versions thereof require significant manipulation in order to generate the fiber form of the molecule. Furthermore, unlike other sources of cellulose, for example from plant and wood sources as well as other sources of chitin and chitosan, the microbially produced copolymers of the present invention are essentially pure polysaccharide. The copolymers of the present invention are easily purified to remove bacterial contaminants using methods well known in the art and as described herein.
The method to generate novel copolymers (cellulose-chitin, cellulose-chitosan) provided herein ameliorates the inherent difficulties associated with modifying plant-derived cellulose. In addition, this direct one-step process offers a simpler and less xe2x80x98environmentally-damagingxe2x80x99 approach than purification and modification of chitin (in terms of organic solvents, heavy metals, catalyst) towards new useful degradable polymers. Additional benefits for these fibrils are facile coupling chemistries that can be used to functionalize the amine groups in the cellulose-chitosan cellulose:chitin or cellulose:chitin:chitosan copolymers. These copolymers are useful for cross-linking, coupling of dyes or pharmaceuticals, or other surface treatments. In addition, chitin and chitosan are useful as relating agents (Muzzarelli, R. A. A., Chitin pp. 207-253, Pergamon Press, New York (1977)), drug carriers (Nakatsuka, S. and Andrady, A. L., J. Appl. Polym. Sci., 44:17-28 (1992)), membranes (Blair, H. S. et al., J. Appl. Polym. Sci., 33:641-656 (1987)), water treatment additives (Asano, T., xe2x80x9cChitosan Applications in Wastewater Sludge Treatment,xe2x80x9d In: Proceedings of the First International Conference on Chitin/Chitosan, R. A. A. Muzzarelli and E. R. Pariser (eds). pp231-252, MIT, Cambridge, Mass., USA (1978)), and wound-healing agents (Austin, P. R. et al., Science, 212:749-753 (1981); Minami, S. et al., Carbohydr. Polym., 29:295-299 (1996)). Furthermore, the potential to incorporate blocks of chitin and chitosan into cellulose as described herein, provides new opportunities for cellulose-derived biomaterials, since the polymers now may be susceptible to lysozyme in the human body.
The present invention is drawn to a method for producing polysaccharide copolymers such as glucose:glucose analog polymers using a biosynthetic agent. The method of the present invention results in production of copolymers wherein the glucose analog is present in said polymer at a mole percent of at least about 1 to about 90 percent.
The present invention is further drawn to polysaccharide copolymers such as glucose:glucose analog polymers comprising glucosamine and glucose wherein glucosamine is present at a mole percent of at least about 1 to about 90 percent. The present invention is further drawn to glucose:glucose analog polymers comprising N-acetylglucosamine and glucose wherein N-acetylglucosamine is present at a mole percent of at least about 7 to about 90 percent. The present invention is further drawn to glucose:glucose analog polymers comprising glucosamine, N-acetylglucosamine and glucose, wherein the glucose analogs are present in the copolymer at a mole percent of at least about 1 to about 90 percent. The formation of chitin-like and chitosan-like polymers by direct bacterial incorporation of N-acetylglucosamine and glucosamine, respectively, as described herein provides new options in the synthesis and purification of consistent materials. This process results in more consistent polymer structural features, such as consistent levels of glucosamine or N-acetylglucosamine incorporation. In addition, the option to control monomer composition within the cellulose-chitin, cellulose-chitosan or cellulose-chitin-chitosan copolymers of the present invention provides new options in tailoring polymer functional properties such as solubility and reactivity. Novel combinations of properties can be envisioned by generating these types of copolymers, versus the individual homopolymer or traditional blends of two of the homopolymers.
Other cellulose-producing bacteria can be used in the method of the present invention. For example, other bacteria such as Agrobacterium tumefaciens, Sarcina ventriculi, or Rhizobium leguminosarum (trifolii) can be used in the method of the present invention. These other microbial sources of cellulose and cellulose:chitin or cellulose:chitosan copolymers are important because the ultrastructure of cellulose fibrils is different when generated from different bacteria. Ribbons and fibrils are formed by A. xylinum, while simple bundles and flocs are generated by A. tumefaciens. These differences may be important in the modulation of functional properties, degree of crystallinity. Also, S. vertriculi, as a Gram-positive cellulose-producing microorganism, generates cellulose that is closely associated with the cell wall and forms cell packets with a fibrillar structure. Furthermore, cellulose II (antiparallel arrangement of individual chains in crystalline cellulose, characteristic of industrially mercerized cellulose) is produced by S. ventriculi, while cellulose I (parallel arrangement of individual chains in crystalline cellulose) is formed by A. xylinum and A. tumefaciens. The ability to generate different copolymers in different chain configurations would be an additional benefit for the industrial and research use of the polymers.